Degree and site of chromosomal instability define its oncogenic potential

Aneuploidy, resulting from chromosomal instability (CIN), is a hallmark of most human solid tumors and correlates with aggressive disease, therapy resistance, and poor prognosis. Despite these associations, whether and how CIN drives tumor initiation and progression has remained unresolved due to conflicting evidence from animal models. Prior mouse studies have variably reported that CIN is neutral, tumor-promoting, or even tumor-suppressive, with comparisons complicated by differing genetic backgrounds, tumor models, and methods to induce CIN. Technical limitations have also hindered direct measurement of CIN levels in relevant tissues, obscuring dose–response relationships. This study addresses the central question of how the degree of CIN and the tissue site determine oncogenic potential. The authors developed a genetic mouse model enabling spatiotemporal, graded induction of CIN and used it to test the hypothesis that specific CIN levels are sufficient to initiate intestinal tumorigenesis and that the impact of CIN on tumor formation differs between small intestine and colon.

CIN is prevalent in 70–90% of solid tumors and is linked to intra-tumoral heterogeneity, immune evasion, metastasis, therapy resistance, and poor prognosis in multiple cancers, including colorectal cancer (CRC). In CRC, CIN tumors (often inferred via aneuploidy) are associated with worse response to 5-fluorouracil and reduced progression-free survival. However, mouse models assessing CIN as a driver of tumor formation have yielded mixed results: spontaneous tumor development is often sporadic and delayed, and in tumor-prone backgrounds CIN has been reported to be neutral, promoting, or suppressive depending on the model. Differences in genetic backgrounds, tumor initiation mechanisms, and non-physiologic methods to induce CIN have precluded clear conclusions, and CIN levels were often not directly quantified in vivo. These gaps motivate a model that can induce and measure defined CIN levels within specific tissues to clarify dose- and tissue-dependent oncogenic effects.

Model generation and CIN induction: The authors engineered a conditional allelic series in the spindle assembly checkpoint kinase Mps1, introducing T649A (TA; hypomorphic) and D637A (KD; kinase-dead) mutations to generate graded reductions in Mps1 activity and thus graded CIN levels (CINki alleles). Recombination was driven by Rosa26-CreER-T2 (systemic, tamoxifen-inducible) or Villin-Cre (intestinal epithelium-specific, developmental induction >12.5 dpc). Mouse embryonic fibroblasts (MEFs) and intestinal organoids were used to characterize CIN levels in vitro. Validation of graded CIN: After 4-hydroxytamoxifen (4-OHT) induction in CINki; Rosa26-CreER-T2 MEFs, recombination and mutant Mps1 expression were confirmed by PCR and Western blot. Reduced Mps1 activity was verified via reduced mitotic phosphorylation and diminished MAD1 kinetochore localization. Time-lapse microscopy quantified mitotic duration and missegregation frequencies, establishing a graded increase in chromosome segregation errors with decreasing Mps1 activity. In vivo, intestinal anaphase figures were analyzed, and single-cell karyotype sequencing (scKaryo-seq) of small intestine one week post induction quantified aneuploidy and karyotype heterogeneity. Tumor assays: For early spontaneous tumorigenesis, intestinal-specific CIN was induced in CINki; Villin-Cre mice. Whole-mount methylene blue staining of intestines at 12 weeks quantified lesions; histology (H&E) confirmed adenomas. Nuclear β-catenin immunohistochemistry assessed Wnt pathway activation in lesions. In tumor-prone settings, CIN was induced in ApcMin/+; Villin-Cre background to assess effects on adenoma burden in small intestine and colon (including distal colon). Distribution and counts of adenomas were performed on whole mounts and corroborated by histology. Proliferation and apoptosis analyses: At ~4 weeks (around adenoma initiation), Ki67 immunostaining quantified proliferative compartment size and proliferative index in crypts from small intestine and colon. Mitotic cells (phospho-Histone H3) and apoptotic bodies (morphology-based) were assessed in crypts to evaluate cell turnover and viability. Organoid live imaging: Small intestine and colon organoids (including from ApcMin/+ backgrounds and adenomas) underwent time-lapse imaging to measure chromosome missegregation frequencies and compare CIN levels across genotypes and tissues. Genomic analyses: scKaryo-seq of normal tissue and adenomas measured copy number changes, aneuploidy burden, and karyotype heterogeneity. Loss of heterozygosity (LOH) at Apc in adenomas was evaluated, including evidence for disomy of mutant chromosome 18. Statistics: Sample sizes were informed by power analysis. Groups were assigned by genotype (no randomization). Statistical tests included one-tailed Student’s t-tests, Welch’s t-tests, and one-way ANOVA with Fisher’s LSD as described in figure legends. Significance thresholds were reported (e.g., p < 0.05, **** for p < 0.0001).

- The CINki allelic series in Mps1 produced graded reductions in spindle assembly checkpoint activity and corresponding graded increases in chromosome missegregation in MEFs, organoids, and in vivo intestinal cells. scKaryo-seq confirmed increased aneuploidy and karyotype heterogeneity in intestinal tissues even one week post induction. - Moderate CIN was sufficient to drive very early onset spontaneous intestinal adenoma formation. In CINki; Villin-Cre mice, increased small intestinal adenomas were detected by 12 weeks of age; lesions showed nuclear β-catenin, indicating Wnt pathway activation. Quantification of lesions showed significant increases with moderate CIN (e.g., number of lesions, p = 0.006 in one comparison; other analyses reported p = 0.002 and p = 0.013 depending on group). - In ApcMin/+ mice, moderate CIN dramatically increased adenoma burden across the intestinal tract and particularly in the distal colon, more closely mirroring human FAP/CRC distribution than ApcMin/+ alone. - A higher CIN level further elevated adenoma formation in the distal colon but did not increase adenomas in the small intestine, revealing a tissue-dependent response to CIN degree. - Moderate and high CIN expanded the proliferative compartment size in colon crypts (at ~4 weeks), but not in small intestine, without changing the proliferative index within the compartment. This suggests enhanced retention of proliferating cells in colon crypts, increasing the likelihood of neoplastic progression in colon. - Adenomas arising under moderate/high CIN were aneuploid and karyotypically heterogeneous. LOH at Apc occurred via disomy of the mutant chromosome 18 rather than simple loss of the wild-type allele, consistent with CIN-facilitated mechanisms of LOH. - Overall, CIN is potently oncogenic; its tumor-promoting effects depend on both the degree of CIN and the tissue context, with moderate CIN often exhibiting maximal oncogenicity in the intestine.

The study directly addresses how the degree and tissue site of CIN determine oncogenic outcomes. By enabling precise, tissue-specific induction of graded CIN, the authors demonstrate that moderate to high CIN is sufficient to initiate intestinal tumorigenesis early in life. Importantly, the relationship between CIN level and tumorigenesis is non-linear and tissue-dependent: moderate CIN strongly promotes adenomas in both small intestine and colon, while higher CIN further increases colon adenomas but has little effect on small intestine. These findings challenge simplified models in which high CIN uniformly suppresses tumorigenesis; instead, the data indicate that certain CIN ranges are maximally tumorigenic and that similar CIN levels can have contrasting effects across tissues. Mechanistically, increased proliferative compartment size in colon (but not small intestine) under moderate/high CIN suggests that CIN may enhance retention and expansion of proliferating cells within colon crypts, facilitating clonal evolution. The observation of aneuploid, heterogeneous adenomas and a CIN-consistent LOH mechanism for Apc supports a model in which CIN both initiates and promotes tumorigenesis, potentially accelerating LOH and enabling selection for advantageous karyotypes. The improved recapitulation of human disease patterns (e.g., distal colon adenomas in ApcMin/+ with CIN) underscores the clinical relevance of the model and indicates that tissue-specific factors modulate the oncogenic impact of CIN. Collectively, the data argue that CIN’s oncogenic potential is governed by a combination of CIN degree and tissue-specific microenvironmental or cellular contexts, with implications for understanding CRC initiation and for designing interventions that consider CIN levels and tissue site.

This work introduces a versatile mouse model that enables spatiotemporal induction of defined CIN levels, allowing direct comparison of dose–response relationships across tissues. The authors show that CIN can be a potent driver of early intestinal adenoma formation, with moderate CIN often exerting maximal oncogenic effects. Critically, the same CIN levels can have divergent impacts across tissues: higher CIN further promotes colon tumorigenesis but does not enhance small intestinal tumor burden. The model better mirrors human CRC/FAP distribution when CIN is layered onto ApcMin/+, highlighting translational relevance. Future research should dissect the mechanisms underlying tissue-specific responses to CIN (e.g., differences in crypt dynamics, stem cell niches, immune interactions), elucidate the precise pathways by which CIN accelerates LOH and karyotypic evolution, and extend the graded CIN approach to other organs and cancer predisposition contexts to map tissue- and dose-dependent oncogenic landscapes.

- While CIN levels were carefully titrated and validated, some organoid and tissue measurements indicated that absolute CIN levels may differ between small intestine and colon, complicating direct comparisons, although key genotypes with different tumor phenotypes showed comparable CIN levels. - In early analyses of 12-week-old intestines, no significant differences were observed in certain viability metrics (e.g., apoptotic bodies per crypt) between moderate and higher CIN, suggesting additional, unmeasured factors may influence tumor initiation. - The precise mechanism of Apc LOH in CIN-driven adenomas (e.g., recombination vs. chromosome missegregation leading to disomy of the mutant allele) remains unresolved. - Animals were assigned to groups by genotype rather than randomized, and some group sizes for particular genotypes/time points were limited, potentially affecting power for certain comparisons. - The study focuses on intestinal epithelium; extrapolation to other tissues will require additional modeling, despite the model’s capability for tissue-specific CIN induction.